Despite advances in ovarian cancer (OC) therapy, recurrent OC remains a poor-prognosis disease. Because of the close interaction between OC cells and the tumor microenvironment (TME), it is important to develop strategies that target tumor cells and engage components of the TME. A major obstacle in the development of OC therapies is the identification of targets with expression limited to tumor surface to avoid off-target interactions. The follicle-stimulating hormone receptor (FSHR) has selective expression on ovarian granulosa cells and is expressed on 50%–70% of serous OCs. We generated mAbs targeting the external domain of FSHR using in vivo–expressed FSHR vector. By high-throughput flow analysis, we identified multiple clones and downselected D2AP11, a potent FSHR surface–targeted mAb. D2AP11 identifies important OC cell lines derived from tumors with different mutations, including BRCA1/2, and lines resistant to a wide range of therapies. We used D2AP11 to develop a bispecific T cell engager. In vitro addition of PBMCs and T cells to D2AP11-TCE induced specific and potent killing of different genetic and immune escape OC lines, with EC50s in the ng/ml range, and attenuated tumor burden in OC-challenged mouse models. These studies demonstrate the potential utility of biologics targeting FSHR for OC and perhaps other FSHR-positive cancers.
Devivasha Bordoloi, Pratik S. Bhojnagarwala, Alfredo Perales-Puchalt, Abhijeet J. Kulkarni, Xizhou Zhu, Kevin Liaw, Ryan P. O’Connell, Daniel H. Park, Daniel W. Kulp, Rugang Zhang, David B. Weiner
The aberrant activation of STAT3 is associated with the etiology and progression in a variety of malignant epithelial-derived tumors, including head and neck squamous cell carcinoma (HNSCC) and colorectal cancer (CRC). Due to the lack of an enzymatic catalytic site or a ligand-binding pocket, there are no small-molecule inhibitors directly targeting STAT3 that have been approved for clinical translation. Emerging proteolysis targeting chimeric (PROTAC) technology–based approach represents a potential strategy to overcome the limitations of conventional inhibitors and inhibit activation of STAT3 and downstream genes. In this study, the heterobifunctional small-molecule–based PROTACs are successfully prepared from toosendanin (TSN), with 1 portion binding to STAT3 and the other portion binding to an E3 ubiquitin ligase. The optimized lead PROTAC (TSM-1) exhibits superior selectivity, potency, and robust antitumor effects in STAT3-dependent HNSCC and CRC — especially in clinically relevant patient-derived xenografts (PDX) and patient-derived organoids (PDO). The following mechanistic investigation identifies the reduced expression of critical downstream STAT3 effectors, through which TSM-1 promotes cell cycle arrest and apoptosis in tumor cells. These findings provide the first demonstration to our knowledge of a successful PROTAC-targeting strategy in STAT3-dependent epithelial cancer.
Jinmei Jin, Yaping Wu, Zeng Zhao, Ye Wu, Yu-dong Zhou, Sanhong Liu, Qingyan Sun, Guizhu Yang, Jiayi Lin, Dale G. Nagle, Jiangjiang Qin, Zhiyuan Zhang, Hong-zhuan Chen, Weidong Zhang, Shuyang Sun, Xin Luan
HIV-specific chimeric antigen receptor–T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell–like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).
Kim Anthony-Gonda, Alex Ray, Hang Su, Yuge Wang, Ying Xiong, Danica Lee, Ariele Block, Vanessa Chilunda, Jessica Weiselberg, Lily Zemelko, Yen Y. Wang, Sarah Kleinsorge-Block, Jane S. Reese, Marcos de Lima, Christina Ochsenbauer, John C. Kappes, Dimiter S. Dimitrov, Rimas Orentas, Steven G. Deeks, Rachel L. Rutishauser, Joan W. Berman, Harris Goldstein, Boro Dropulić
The folding and trafficking of transmembrane glycoproteins are essential for cellular homeostasis and compromised in many diseases. In Niemann-Pick type C disease, a lysosomal disorder characterized by impaired intracellular cholesterol trafficking, the transmembrane glycoprotein NPC1 misfolds due to disease-causing missense mutations. While mutant NPC1 has emerged as a robust target for proteostasis modulators, these drug development efforts have been unsuccessful in mouse models. Here, we demonstrate unexpected differences in trafficking through the medial Golgi between mouse and human I1061T-NPC1, a common disease-causing mutant. We establish that these distinctions are governed by differences in the NPC1 protein sequence rather than by variations in the ER folding environment. Moreover, we demonstrate direct effects of mutant protein trafficking on the response to small molecules that modulate the endoplasmic reticulum folding environment by affecting Ca++ concentration. Finally, we develop a panel of isogenic human NPC1 iNeurons expressing wild type, I1061T-, and R934L-NPC1 and demonstrate their utility in testing these candidate therapeutics. Our findings identify important rules governing mutant NPC1’s response to proteostatic modulators and highlight the importance of species- and mutation-specific responses for therapy development.
Mark L. Schultz, Kylie J. Schache, Ruth D. Azaria, Esmée Q. Kuiper, Steven Erwood, Evgueni A. Ivakine, Nicole Y. Farhat, Forbes D. Porter, Koralege C. Pathmasiri, Stephanie M. Cologna, Michael D. Uhler, Andrew P. Lieberman
Pancreatic neuroendocrine tumors (PNETs) are malignancies arising from the islets of Langerhans. Therapeutic options are limited for the over 50% of patients who present with metastatic disease. We aimed to identify mechanisms to remodel the PNET tumor microenvironment (TME) to ultimately enhance susceptibility to immunotherapy. The TMEs of localized and metastatic PNETs were investigated using an approach that combines RNA-sequencing, cancer and T cell profiling, and pharmacologic perturbations. RNA-sequencing analysis indicated that the primary tumors of metastatic PNETs showed significant activation of inflammatory and immune-related pathways. We determined that metastatic PNETs featured increased numbers of tumor-infiltrating T cells compared to localized tumors. T cells isolated from both localized and metastatic PNETs showed evidence of recruitment and antigen-dependent activation, suggestive of an immune-permissive microenvironment. A computational analysis suggested that vorinostat, a histone deacetylase inhibitor, may perturb the transcriptomic signature of metastatic PNETs. Treatment of PNET cell lines with vorinostat increased chemokine CCR5 expression by NF-κB activation. Vorinostat treatment of patient-derived metastatic PNET tissues augmented recruitment of autologous T cells, which was substantiated in a mouse model of PNET. Pharmacologic induction of chemokine expression may represent a promising approach for enhancing the immunogenicity of metastatic PNET TMEs.
Jacques Greenberg, Jessica Limberg, Akanksha Verma, David Kim, Xiang Chen, Yeon J. Lee, Maureen D. Moore, Timothy M. Ullmann, Jessica W. Thiesmeyer, Zachary Loewenstein, Kevin J. Chen, Caitlin E. Egan, Dessislava Stefanova, Rohan Bareja, Rasa Zarnegar, Brendan M. Finnerty, Theresa Scognamiglio, Yi-Chieh Nancy Du, Olivier Elemento, Thomas J. Fahey III, Irene M. Min
Vogt-Koyanagi-Harada (VKH) disease is an important refractory uveitis mediated by pathological T cells (TCs). Tofacitinib (TOFA) is a Janus kinases (JAKs) targeted therapy for several autoimmune diseases. However, the specific pathogenesis and targeted therapeutics for VKH remain largely unknown. Based on single-cell RNA sequencing and mass cytometry, we present the first multimodal high-dimensional analysis to determine a comprehensive human immune atlas of VKH patients undergoing TOFA therapy in the context of subset composition, gene signatures, enriched pathways, and intercellular interactions. VKH patients are characterized by TCs polarization from naive to effector and memory subsets, altogether with accrued monocytes, upregulated cytokines and JAK-STAT signaling pathways. In vitro, TOFA reversed Th17/ regulatory T-cell (Treg) imbalance and inhibited IL-2-induced STAT1/3 phosphorylation. TOFA alleviated VKH symptoms by restoring pathological TCs polarization and functional marker expression and downregulating cytokine signaling and lymphocyte function. Remarkably, inflammation-related responses and intercellular interactions decreased after TOFA treatment, particularly in monocytes. Notably, we identified two inflammation- and JAK-associated monocyte subpopulations that were strongly implicated in VKH pathogenesis and mechanisms involved in TOFA treatment. Here, we provide a novel JAK-targeted therapy for VKH and elaborate on the possible therapeutic mechanisms of TOFA, expanding our knowledge of VKH pathological patterns.
Xiuxing Liu, Qi Jiang, Jianjie Lv, Shizhao Yang, Zhaohao Huang, Runping Duan, Tianyu Tao, Zhaohuai Li, Rong Ju, Yingfeng Zheng, Wenru Su
The muscular dystrophies (MDs) are genetic muscle diseases that result in progressive muscle degeneration followed by the fibrotic replacement of affected muscles as regenerative processes fail. Therapeutics that specifically address the fibrosis and failed regeneration associated with MDs represent a major unmet clinical need for MD patients, particularly those with advanced-stage disease progression. The current study investigated targeting NAD(P)H oxidase 4 (NOX4) as a potential strategy to reduce fibrosis and promote regeneration in disease-burdened muscle that models Duchenne muscular dystrophy (DMD). NOX4 was elevated in the muscles of dystrophic mice and DMD patients, localizing primarily to interstitial cells located between muscle fibers. Genetic and pharmacological targeting of NOX4 significantly reduced fibrosis in dystrophic respiratory and limb muscles. Mechanistically, NOX4 targeting decreased the number of fibrosis-depositing cells (myofibroblasts) and restored the number of muscle-specific stem cells (satellite cells) localized to their physiological niche, thereby rejuvenating muscle regeneration. Furthermore, acute inhibition of NOX4 was sufficient to induce apoptotic clearing of myofibroblasts within dystrophic muscle. These data indicate that targeting NOX4 is an effective strategy to promote the beneficial remodeling of disease-burdened muscle representative of DMD and, potentially, other MDs and muscle pathologies.
David W. Hammers
Recessive PJVK mutations that cause a deficiency of pejvakin, a protein expressed in both sensory hair cells and first-order neurons of the inner ear, are an important cause of hereditary hearing impairment. Patients with PJVK mutations garner limited benefits from cochlear implantation; thus, alternative biological therapies may be required to address this clinical difficulty. The synthetic adeno-associated viral vector Anc80L65, with its wide tropism and high transduction efficiency in various inner ear cells, may provide a solution. We delivered the PJVK transgene to the inner ear of Pjvk mutant mice using the synthetic Anc80L65 vector. We observed robust exogenous pejvakin expression in the hair cells and neurons of the cochlea and vestibular organs. Subsequent morphologic and audiologic studies demonstrated significant restoration of spiral ganglion neuron density and hair cells in the cochlea, along with partial recovery of sensorineural hearing impairment. In addition, we observed a recovery of vestibular ganglion neurons and balance function to WT levels. Our study demonstrates the utility of Anc80L65-mediated gene delivery in Pjvk mutant mice and provides insights into the potential of gene therapy for PJVK-related inner ear deficits.
Ying-Chang Lu, Yi-Hsiu Tsai, Yen-Huei Chan, Chin-Ju Hu, Chun-Ying Huang, Ru Xiao, Chuan-Jen Hsu, Luk H. Vandenberghe, Chen-Chi Wu, Yen-Fu Cheng
Deficiency of glycogen debranching enzyme in glycogen storage disease type III (GSD III) results in excessive glycogen accumulation in multiple tissues, primarily the liver, heart, and skeletal muscle. We recently reported that an adeno-associated virus (AAV) vector expressing a bacterial debranching enzyme (Pullulanase) driven by the ubiquitous CMV enhancer/chicken β-actin (CB) promoter cleared glycogen in major affected tissues of infant GSD IIIa mice. In this study, we developed a novel dual promoter consisting of a liver-specific promoter (LSP) and the CB promoter for gene therapy in adult GSD IIIa mice. Ten-week treatment with an AAV vector containing the LSP-CB dual promoter in adult GSD IIIa mice significantly increased Pullulanase expression and reduced glycogen contents in the liver (-60%), heart (-76%), and skeletal muscle (-63%), accompanied by the reversal of liver fibrosis, improved muscle function, and significant decrease in plasma biomarkers alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Compared to the CB promoter, the dual promoter effectively decreased Pullulanase-induced cytotoxic T lymphocyte responses and enabled persistent therapeutic gene expression in adult GSD IIIa mice. Future studies are needed to determine the long-term durability of the dual promoter mediated expression of Pullulanase in adult GSD IIIa mice and in large animal models.
Jeong-A Lim, Priya S. Kishnani, Baodong Sun
BACKGROUND. A patient-derived organoid (PDO) platform may serve as a promising tool for translational cancer research. In this study, we evaluated PDO’s ability to predict clinical response to gastrointestinal (GI) cancers. METHODS. We generated PDOs from primary and metastatic lesions of patients with GI cancers, including pancreatic ductal adenocarcinoma, colorectal adenocarcinoma, and cholangiocarcinoma. We compared PDO response with the observed clinical response for donor patients to the same treatments. RESULTS. We reported an approximately 80% concordance rate between PDO and donor tumor response. Importantly, we found a profound influence of culture media on PDO phenotype, where we showed significant difference in response to standard of care chemotherapies, distinct morphologies, and transcriptomes between media within the same PDOs cultures. CONCLUSION. While we demonstrate a high concordance rate between donor tumor and PDO, these studies also showed the important role of culture media when using PDOs to inform treatment selection and predict response across a spectrum of GI cancers. TRIAL REGISTRATION. Not applicable. FUNDING. This work was supported by the Joan F. & Richard A. Abdoo Family Fund in Colorectal Cancer Research, CA265050, GI Cancer program of the Mayo Clinic Cancer Center, Mayo Clinic SPORE in Pancreatic Cancer, Center of Individualized Medicine (Mayo Clinic), Department of Laboratory Medicine and Pathology (Mayo Clinic), Incyte Pharmaceuticals and Mayo Clinic Hepatobiliary SPORE, a University of Minnesota-Mayo Clinic Partnership grant, and the Early Therapeutic program (Department of Oncology, Mayo Clinic).
Tara L. Hogenson, Hao Xie, William J. Phillips, Merih D. Toruner, Jenny J. Li, Isaac P. Horn, Devin J. Kennedy, Luciana L. Almada, David L. Marks, Ryan M. Carr, Murat Toruner, Ashley N. Sigafoos, Amanda N. Koenig-Kappes, Rachel L.O. Olson, Ezequiel J. Tolosa, Cheng Zhang, Hu Li, Jason D. Doles, Jonathan Bleeker, Michael T. Barrett, James H. Boyum, Benjamin R. Kipp, Amit Mahipal, Joleen M. Hubbard, Temperance J. Scheffler Hanson, Gloria M. Petersen, Surendra Dasari, Ann L. Oberg, Mark J. Truty, Rondell P. Graham, Michael J. Levy, Mojun Zhu, Daniel D. Billadeau, Alex A. Adjei, Nelson Dusetti, Juan L. Iovanna, Tanios S. Bekaii-Saab, Wen Wee Ma, Martin E. Fernandez-Zapico
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